Worldwide, hepatitis B virus (HBV) infection is the most common viral hepatitis having infected over two billion people and chronically infecting more than 400 million, putting them at increased risk to develop cirrhosis and hepatocellular carcinoma. In the United States over one million people have chronic hepatitis B infection. Clinical therapy is targeted to the suppression of viral replication but the virus is able to persit in a nonreplicative covalently closed circular form called cccDNA, with the potential to reactivate upon immune suppression or with aging. As a consequence, the hepatitis B virus in chronic HBV infections is challenging to eradicate and cure is rare. The difficulty in developing new HBV therapies has been due to the lack of good model systems. The current model system to study hepatitis B has been hepatoma cell lines in which HBV is over expressed transiently or stably to produce all viral gene products and maintain replication. However such models over express HBV in a non-physiological manner and do not faithfully recapitulate adult hepatocyte phenotype or function as they have undergone a variety of genetic and metabolic changes. As a consequence, the viral entry factors as well as the regulatory processes of viral replication have been poorly understood. This has prevented development of therapeutic strategies that achieve viral control and eradication. This proposal builds on new tools and techniques that enable the long-term culture of metabolically functional primary human hepatocytes and the establishment of pluripotent stem cell derived hepatocyte-like cells. Both innovations enables the exploration of the determinants of HBV entry, viral replication, and the cccDNA state which will open new opportunities to better understand HBV biology and develop new targeted therapies. The establishment of a culture model system that is permissive for HBV infection and also enables the long-term culture of metabolically functional primary human hepatocytes represents a new opportunity to explore the determinants of HBV entry, viral replication, and cccDNA formation and persistence (Aims 1-2). In contrast to current hepatoma cell lines, a functional hepatocyte screening platform will enable the study of the role that biologic pathways play in the HBV viral life cycle's choice towards cccDNA production versus active HBV virion production (Aim 2). These pathways can then be explored and dissected in detail in induced pluripotent derived stem cell derived variants to determine their impact on hepatocyte permissiveness, hepatitis B virus pathogenesis and cccDNA formation (Aim 3). Better insights in HBV pathogenesis, cccDNA formation, virus evasion and the determinants of hepatocyte autonomous responses may also be translational as treatment options for chronically infected HBV patients are available but are unable to eliminate cccDNA leading to lifelong infection and requirements for lifelong treatment.